Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

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Molecular Biology of the CellVol. 17, 1239–1249, March 2006

Rapid, Diffusional Shuttling of Poly(A) RNA betweenNuclear Speckles and the Nucleoplasm□D

Joan C. Ritland Politz,* Richard A. Tuft,† Kannanganattu V. Prasanth,‡Nina Baudendistel,§ Kevin E. Fogarty,† Larry M. Lifshitz,† Jorg Langowski,§David L. Spector,‡ and Thoru Pederson*

*Department of Biochemistry and Molecular Pharmacology and Program in Cell Dynamics and †Departmentof Physiology, University of Massachusetts Medical School, Worcester, MA 01605; ‡Cold Spring HarborLaboratory, Cold Spring Harbor, NY 11724; and §German Cancer Research Center, 69120 Heidelberg,Germany

Submitted October 14, 2005; Accepted December 2, 2005Monitoring Editor: A. Gregory Matera

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A)RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles isdynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLacells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescencecorrelation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in bothspeckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in whichcaged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckleor nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the taggedpoly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless ofwhether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactiva-tion-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNAin either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not requiremetabolic energy.

INTRODUCTION

Nuclear speckles are morphologically distinct regions of thenucleoplasm that contain pre-mRNA splicing componentsas well as poly(A) RNA (Carter et al., 1993; Zhang et al., 1994;Lamond and Spector, 2003). They are operationally definedby their immunostaining with a variety of pre-mRNA splic-ing factor antibodies, and they also show in situ hybridiza-tion signal using probes for poly(A). When these sites areviewed in the electron microscope, most of them are foundto represent interchromatin granule clusters (Fakan et al.,1984, Deerinck et al., 1994). However, RNA polymerase IItranscription sites are distributed throughout the nucleus,indicating that although some of the RNA present in inter-chromatin granules/speckles is nascent, transcription is notrestricted to this compartment, and much of the poly(A)RNA there has likely been transcribed previously and/or atdistant sites (Fakan and Nobis, 1978; Wansink et al., 1993,

Zeng et al., 1997, Neugebauer and Roth, 1997, Cmarko et al.,1999; Guillot et al., 2004). Indeed, although transcripts thatare being rapidly produced or that contain many introns aresometimes observed in speckles at the light microscopy level(Johnson et al., 2000; Shopland et al., 2003), the majority ofstudies over the years has indicated that most speckles arenot primary sites of pre-mRNA splicing (for reviews, seeMattaj, 1994; Huang and Spector, 1996a; Neugebauer andRoth, 1997; Lamond and Spector, 2003). In two cases, a singlespecies of pre-mRNA has been followed from its transcrip-tion site to the nuclear pore, and in neither case did the RNAseem to accumulate in nuclear subcompartments (Singh etal., 1999; Shav-Tal et al., 2004). Instead, it seems that splicingcomponents typically move away from speckles to sites ofgene expression where splicing occurs cotranscriptionally(Huang and Spector, 1996b; Misteli et al., 1997, 1998; Eils etal., 2000). These findings have therefore made it difficult tounderstand why poly(A) RNA is associated with speckles.

One idea has been that perhaps there is a metabolicallystable, nucleus-restricted poly(A) RNA population thathelps to organize and structurally define certain nuclearbodies such as speckles (Fakan et al., 1984; Huang et al., 1994;Lamond and Spector, 2003). This idea stems from observa-tions that as much as 30% of the poly(A) RNA never leavesthe nucleus in growing mammalian cells (Perry et al., 1974,Herman et al., 1976). At least some of this poly(A) RNAconsists of noncoding sequences that seem to have a varietyof nuclear functions (Morey and Avner, 2004). We earlierstudied the movement of poly(A) RNA in the nucleus of live

This article was published online ahead of print in MBC in Press(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–10–0952)on December 21, 2005.□D The online version of this article contains supplemental materialat MBC Online (http://www.molbiolcell.org).

Address correspondence to: J.C.R. Politz (joan.politz@umassmed.edu).

Abbreviations used: FCS, fluorescence correlation spectroscopy;mRFP-SC35, monomeric red fluorescent protein fused to SC35 pro-tein; oligo, oligodeoxynucleotide.

rat myoblasts using the technique of fluorescence correlationspectroscopy (FCS) (Politz et al., 1998) and found that al-though there was a class of poly(A) RNA that moved ratherrapidly throughout the nucleoplasm, there also was a sizablefraction that moved much more slowly. It was thereforepossible that this fraction represented molecules that wereconfined within nuclear bodies such as speckles and thusconstrained in their motion.

In the present investigation, we developed methods toaddress the possible structural nature of the poly(A) RNAdirectly in the nuclear speckles of living cells. A stable cellline expressing a chimeric SC35 protein coupled to a mono-meric red fluorescent protein (mRFP-SC35) was generated,and the chimeric protein was found to behave similarly tothe native SC35 protein (Politz et al., 2003). Endogenousnuclear poly(A) RNA was tagged in these cells using fluo-rescently labeled oligo(dT) as a hybridization probe (Peder-son, 1999; Politz, 1999; Politz et al., 1999). We used both FCS(Magde et al., 1972; Politz et al., 1998; Wachsmuth et al., 2000)and photoactivation-tracking techniques (Politz, 1999; Politzet al., 1999, 2003, 2004) to measure the mobility of the taggedpoly(A) RNA inside speckles and in the nucleoplasm. Time-lapse digital imaging microscopy was also used to visuallytrack the movement of photoactivated tagged poly(A) RNAas it moved between speckles and the nucleoplasm. Weshow that poly(A) RNA moves in and out of speckles withthe characteristics of a diffusive process and that there is nota substantial immobile population of poly(A) RNA withinthe speckle as might be expected if it were serving as astructural scaffolding. In fact, the behavior of poly(A) RNAwithin and outside speckles was indistinguishable, and therate of movement did not change when the temperature waslowered from 37 to 22°C. The results indicate that ratherthan speckles harboring an actively sequestered, kineticallydistinct population, the poly(A) RNA that is associated witha speckle at any given time exchanges freely with the nucle-oplasm and other speckles.

MATERIALS AND METHODS

mRFP-SC35 Stable Cell Line and TransfectionPCR was used to generate a restriction site at the stop codon of a human SC35cDNA (Prasanth et al., 2003) for cloning into a vector encoding the mRFP(Campbell et al., 2002). A HeLa stable cell line containing the mRFP-SC35 wasgenerated and maintained in DMEM (low glucose) with 10% fetal bovineserum (FBS) and 0.5 mg/ml G418 (Invitrogen, Carlsbad, CA).

�-Tropomyosin Mini-Gene Transfection and In SituHybridizationElectroporation was performed on trypsinized cells (240 V; 950 �F), whichwere then resuspended in 250 �l of growth medium and incubated with 4 �gof a rat �-tropomyosin mini-gene construct (Helfman et al., 1988) plus 20 �gof sheared salmon sperm DNA. Cells were then seeded onto acid-washedcoverslips and processed for in situ hybridization 24 h posttransfection. The�-tropmyosin probe was labeled with Spectrum-Green-dUTP using a nicktranslation reagent kit (Vysis, Downer’s Grove, IL), and in situ hybridizationwas performed essentially as described previously (Huang and Spector,1996b). Cells were hybridized with 100 ng of probe in 20 �l of hybridizationmixture (50% formamide, 2� SSC, 5% dextran sulfate, and 20 �g of yeasttRNA) at 37°C for 12–14 h. After posthybridization washes, DNA was stainedwith 4�-6-diamidino-2-phenylindole. Imaging acquisitions for in situ experi-ments were performed with a Zeiss Axioplan microscope and a 100� oilimmersion objective. Images were processed using Openlab software (Impro-vision, Lexington, MA).

Immunoblot AnalysisCell lysates were prepared from the mRFP-SC35 stable cell line and treated for30 min at 37°C with 500 U/ml calf intestinal phosphatase (CIP; New EnglandBiolabs, Beverly, MA), and, after gel electrophoresis, were blotted onto anitrocellulose membrane and probed by an antibody that recognizes mRFP(Chemicon International, Temecula, CA).

Oligodeoxynucleotide (Oligo) SynthesisOligo(dT) and control oligo(dA) probes were synthesized by Integrated DNATechnologies (Coralville, IA) as 43mers, with a thymidine containing a C6-aminohexyl group present at every 10 bases [in both oligo(dT) and oligo(dA)].The oligos were then labeled with either fluorescein (Molecular Probes,Eugene, OR) or caged fluorescein (Mitchison et al., 1994) as described previ-ously (Politz et al., 1999, 2004). Both the aminohexyl linker arms and thespacing of the labeling groups along the oligo are thought to prevent RNaseH degradation of the RNA target after the oligo is hybridized (Ueno et al.,1997).

Oligo Uptake and Live Cell ImagingCells were either plated into two-well dishes (Nalge Nunc, Naperville, IL) oronto 25-mm coverslips and transfected with oligo(dT) or oligo(dA) as de-scribed previously (Politz et al., 2004) except that the concentration of oligowas 0.125 �M. After a 1-h incubation without oligo in DMEM (with serum),cells on coverslips were mounted in a holder and then maintained at 37 or22°C as described in DMEM buffered with 25 mM HEPES (10% FBS, nophenol red) (Politz et al., 2004). Cells containing fluorescein-labeled oligo(dT)growing in two-well dishes were imaged using a Quantix 57 charge-coupleddevice camera (Roper Scientific Photometrics, Tucson, AZ) coupled to a LeicaDMIRB microscope equipped with a 100� objective (numerical aperture 1.4)as described previously (Politz et al., 2000). Signal intensity was quantitativelyscaled using MetaMorph software. All uncaging, rapid acquisition digitalmicroscopy, and image processing were performed using equipment andsoftware previously described in detail (Politz et al., 1999, 2004). Some imageswere deconvolved using exhaustive photon reassignment (Carrington et al.,1995).

In Situ Reverse TranscriptionCells were allowed to take up oligo as described above (at an oligo concen-tration of 0.5 �M in the medium) and after a 1-h efflux period, fixed andsubjected to in situ reverse transcription as described in detail previously(Politz et al., 1995; Politz and Singer, 1999), except that the anti-digoxigeninantibodies were linked to horseradish peroxidase (anti-digoxigenin-POD, Fabfragments; Roche Diagnostics, Indianapolis, IN), and the blocking step wasperformed using 1% normal sheep serum (The Jackson Laboratory, Bar Har-bor, ME) in SSC. The colorimetric assay was carried out using a diaminoben-zidine substrate according manufacturer’s instructions (Roche Diagnostics),and bright field images of the resulting signal were captured using the Leicamicroscope system described above. Contrast was quantitatively scaled usingMetaMorph software (Universal Imaging, Downingtown, PA).

Fluorescence Correlation SpectroscopyFCS (Magde et al., 1972) was carried out using a previously described instru-ment (Wachsmuth et al., 2003). Cells were grown in eight-well dishes (NalgeNunc) in RPMI medium (with 10% serum, no phenol red) and transfected asdescribed above. The dishes were then mounted on the stage of the FCSmicroscope, and either a speckle or a site in the nucleoplasm was aligned inthe laser path using a galvanometer scanner in point-addressable mode.Measurements were recorded at a laser intensity of �1–5 kW/cm2 (0.1–0.5-mW laser power at the focus). A 485-nm excitation filter (DF 22; OmegaOptical, Brattleboro, VT) was placed in the laser light path to reduce theintensity of the mRFP for imaging of the fluorescein-labeled oligo. For exper-iments done at 37°C, a chamber around the microscope and stage was heatedto maintain a temperature of 37°C in a humidified 5% CO2 atmosphere. Tenreadings were taken at each site and averaged; readings that showed bleach-ing were discarded. Best fits for the autocorrelation curves were chosen andrecorded using Quickfit (Press et al., 1992), and in some cases, the MaxEntfitting program (Modos et al., 2004) was also used to help determine which fitwas optimal.

SimulationsA 100-�m aperture placed in the UV illumination path during photoactiva-tion produced an �1-�m uncaging spot at the focal plane. This spot is theconvolution of the three-dimensional point-spread-function (3-D PSF) of themicroscope with the two-dimensional (2-D) illumination aperture. In wide-field imaging systems, the captured images of fluorescence are the result ofthe convolution of the 3-D fluorescent oligo distribution with this 3-D PSF. Togenerate computer simulations of the uncaging and imaging processes, the3-D PSF of the microscope system was measured using subresolution fluo-rescent beads (Carrington et al., 1995). Images spanning � 10 �m of focusabout the bead were acquired at 0.25-�m intervals, with a pixel size of 0.15�m to match the experimental data. Custom software designed for modelingreaction and diffusion events inside cells in 3-D (Zou et al., 1999; Fogarty et al.,2000; ZhuGe et al., 2000) was then used to simulate both the uncaging anddiffusion of the fluorescent oligos. The nucleus was modeled as a cylinderwith its central axis along the z-axis (axis of focus) of the microscope (x,y � 15�m; z � 10 �m; pixel size � 0.15 �m). The initial 3-D distribution of uncagedoligo was calculated by digitally convolving a computer generated image ofa 1-�m, 2-D aperture with the empirically measured 3-D PSF and was used as

J.C.R. Politz et al.

Molecular Biology of the Cell1240

the starting distribution for the diffusion simulations. The 3-D uncaged spotwas centered within the cylinder (as necessitated by the imposed symmetry)and focused 4 �m from the bottom. The edges of the nucleus were a barrierto diffusion. Diffusion was simulated using a single diffusion constant, D(units of square micrometers per second), for up to 30 s, and images weresaved at times corresponding to the experimental data. The saved simulationimages were converted from cylindrical coordinates to Cartesian coordinates(x,y,z) using interpolation to produce 3-D images having pixel sizes of 0.15�m in x and y, and 0.25 �m in z. Each 3-D image time point was then“blurred” by convolving with the 3-D PSF and the infocus image was ex-tracted, producing a temporal sequence of 2-D images of diffusion modelingthe experimental data.

Simulations were conducted using a range of diffusion constants. Becausethe microscope system can be approximated as a linear system (Carrington etal., 1995), mixes of two or more different Ds were created by simulating eachD separately. The resulting image sequences for each D were digitally addedin the desired proportions. In the case of modeling oligo confined to a“speckle,” the initial uncaged distribution was masked using a 2-�m-diame-ter sphere center at the infocus position of the spot, and D was set to zero. Forthe simulation shown in Figure 6, D–F, the step convolving the simulationwith the 3-D PSF was omitted.

The spatial profile of the UV uncaging spot intensity at the focal plane,before diffusion, can be well fit with a single Gaussian:

I�x� �A

�2��23/2 ex2

2�2 (1)

where � is the e1 half-width of the spot. The equation for the spatial profileproduced by diffusion of this initial spot is

I�x, t� �1

�2��2 Dt�3/2 ex2

2�2Dt� � Aex2

2�2 (2)

where D is the diffusion constant, t is time, � is as for Eq. 1, and * denotesconvolution. The convolution of two Gaussians is another Gaussian:

I�x, t� �A

�2��2 Dt � �2�3/2 ex2

2�2Dt��2� (3)

We can well fit the spatial profile of the image of the initial uncaged fluores-cence, before diffusion, with the sum of two Gaussian spatial profiles, roughlycorresponding to the infocus and out-of-focus components of the 3-D fluo-rescence distribution:

I�x� � A0ex2

2�02 � A1e

2�12 (4)

and the corresponding diffusion profile:

I�x, t� �A0

�2��2 Dt � �02�3/2 e

2�2Dt��02� �

�2��2 Dt � �12�3/2 e

2�2Dt��12� (5)

The 2-D spatial and temporal intensity profile of both simulated images andexperimental images were fit to either Eq. 3 or Eq. 5, using custom softwareimplementing a multiparameter least-squares curve fit approach, to deriveestimates of both D and �i.

RESULTS

Characterization of Oligo(dT) Uptake by a Stable HeLaCell Line Expressing mRFP-SC35A stable HeLa cell line expressing the splicing factor SC35fused to monomeric red fluorescent protein (mRFP-SC35)was generated using standard procedures (see Materials andMethods). Greater than 95% of the cell population showedexpression of the red SC35 protein, which was observed tobe most concentrated at multiple nucleoplasmic sites (Figure1A). Signal was also observed in nucleoplasmic regions out-side the speckles. A similar distribution pattern has alsobeen observed in stable cell lines expressing the splicingprotein SF2/ASF linked to green fluorescent protein (GFP)(Phair and Misteli, 2000). The transformed cell populationmaintained nearly 100% expression of mRFP-SC35 over sev-eral generations.

We examined the phosphorylation status of the chimericmRFP-SC35 protein and found it to be present in its active,phosphorylated form in the stably transfected cell line (Fig-ure 1B), demonstrating that this mRFP fusion protein wasbehaving similarly to its endogenous SC35 counterpart(Misteli and Spector, 1996). To further test the degree towhich the behavior of the mRFP-SC35 fusion protein resem-bled that of the native SC35 protein, we investigated itsdynamic association with a specific polymerase II transcrip-tion site. As shown in Figure 1C, the mRFP-SC35 chimericprotein was recruited to the sites of active transcription of atransfected �-tropomyosin gene, similar to the behavior ofnative SC35 (Huang and Spector, 1991; Jimenez-Garcia andSpector, 1993; Huang and Spector, 1996b; Misteli et al., 1997;Misteli and Spector, 1999). The cells also seemed morpho-logically normal and grew with a doubling time similar tothe parental cell line. Therefore, by all these criteria themRFP-SC35 fusion protein seemed to be mirroring the be-havior of the native SC35 protein and therefore was judgedto be a valid marker for speckles in these cells.

Introduction of fluorescein-labeled oligo(dT) into this sta-bly transfected HeLa cell line (see Materials and Methods) wasoptimized to give detectable signal in greater than 50% ofthe cells, with minimal effect on cell viability (as judged bydegree of spreading and population size 24 h later; Politz1999; Politz et al., 2004). As previously found in L6 myoblasts(Politz et al., 1995, 1999), oligo(dT) signal was distributedthroughout the nucleus and within speckles but did notseem concentrated in speckles (Figure 2, A and B). An in situreverse transcription assay (Eberwine et al., 1992; Politz and

Figure 1. Characteristics of HeLa cell linestably expressing mRFP-SC35. (A) Raw andrestored (exhaustive photon reassignment;see Materials and Methods) midplanes of anoptical stack showing a live HeLa cell stablyexpressing mRFP-SC35. Bars, 4.2 �m. (B) Im-munoblot of total cellular proteins probedwith RFP antibody before () and after (�)treatment with CIP in wild-type HeLa cells(left lane) and the stable SC35 HeLa cell line(middle and right lanes). (C) Fluorescent insitu hybridization to fixed SC35 stable HeLacell line transiently transfected with a plas-mid that expresses �-tropomyosin mRNA athigh levels (see Materials and Methods). �-Tro-pomyosin mRNA was detected with a Spec-trum-Green-labeled probe (green) and over-lap with mRFP-SC35 (red) is shown asyellow. Bar, 5 �m.

Poly(A) RNA Diffuses through Speckles

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Singer, 1999; Politz et al., 1999) was used to detect hybrid-ization of the oligo to endogenous RNA. In this assay, onlyoligo(dT) that is hybridized to poly(A) can act as a primerfor reverse transcription. As can be seen in Figure 2C, re-verse transcription products were observed in both the nu-cleus and the cytoplasm of cells that took up oligo(dT), asexpected for hybridization to poly(A) RNA. Cells that hadtaken up the control oligo(dA) showed no signal (Figure2D), indicating that the oligo(dA) does not hybridize appre-ciably to RNA in the cell. (HeLa cell nuclear RNA containssome oligo(U) tracts, but these are present at a much lowerconcentration than poly(A); Kish and Pederson, 1977).

FCS Used to Measure Mobility of Poly(A) RNA inSpeckles and the NucleoplasmWe first used the technique of FCS (Magde et al., 1972;Krichevsky and Bonnet, 2002; Kim and Schwille, 2003) tocompare the mobility of poly(A) RNA in speckles and thenucleoplasm. This method quantitatively measures theamount of fluorescence in a very small volume and rapidlymonitors the fluctuation of that signal intensity over time.The more the signal fluctuates, the more rapidly the mole-cules are moving in and out of the interrogated volume. Thisis manifest as an increased rate of decay in the autocorrela-tion function. Using curve-fitting algorithms, the number ofdifferently diffusing components within the sample area canbe determined, and their respective diffusion coefficients

were estimated (Politz et al., 1998; Wachsmuth et al., 2000).The confocal assay volume of FCS is less than a femtoliter; inour experiments, the radial diameter of the confocal volumewas 0.44 �m and the z-axis height was �1.6 �m whenexciting fluorescein (and 0.5 and 2 �m, respectively, whenexciting mRFP). These dimensions are similar to or smallerthan that of a speckle; deconvolved images of the stableSC35 cell line used to estimate speckle size gave radialdiameters of �0.5–2.5 �m and z-axis heights of �1.5–2.5 �m(our unpublished data). Therefore, FCS measurements couldbe taken inside a speckle without substantial excitation ofthe surrounding nucleoplasm.

We used the galvanometer scanner of the fluorescencefluctuation microscope in the point-addressable mode todirect a laser beam into a confocal volume inside the nucleusof live SC35-expressing HeLa cells containing fluorescentoligo(dT). The beam was focused either within a speckle orinto an area of the nucleoplasm devoid of a speckle. Fluo-rescence fluctuations in each interrogated volume were thenmeasured over time and recorded as an autocorrelationcurve. Figure 3A shows representative autocorrelationcurves obtained within (blue) and outside (red) a speckle,with the best fit curves (smooth lines) shown for each in thesame color. It can be seen that very similar autocorrelationcurves were obtained from both speckle and nucleoplasmicsites, indicating that the oligo(dT) had similar moleculardynamics at both sites. Additionally, the inverse particlenumber (the y-intercept), which is proportional to thetotal concentration of oligo(dT), is very similar in bothcases. The best fit curves most often represented two orthree components of differing mobilities. The relative frac-tion of each of these components is shown in Figure 3B(dT on and dT off).

One of these kinetic components was not observed whenmeasurements were made in cells containing the nonhybrid-izing control probe, oligo(dA), and is therefore highly likelyto represent oligo(dT) hybridized to poly(A) RNA (Figure3B, compare red bars in dT columns to the zero-level redbars in the dA columns). No significant difference in themobility of this fraction was observed when measurementswere made either within a speckle or in the nucleoplasm(Figure 3B, compare red fraction in dT off to dT on). It tookthe oligo(dT):poly(A) RNA hybrids an average of 22 � 5 ms(D � 0.65 � 0.19 �m2/s) to traverse the confocal volumewithin a speckle and 20 � 4 ms (D � 0.70 � 0.19 �m2/s) totraverse the same distance in the nucleoplasm. Thus, weobserved no significant difference in mobility of the poly(A)RNA populations inside or outside a speckle.

The fastest moving component present in the oligo(dT)-containing cells (Figure 3B, dT black bars) closely corre-sponded to the most prevalent (�80% of signal) mobilityfraction obtained when measurements were made in controloligo(dA)-containing cells (Figure 3B, dA black bars) andtherefore probably represents unhybridized oligo(dT) in thenucleus (average dT on, 1.1 � 0.2 ms; dT off, 1.1 � 0.3 ms; dAon, 1.8 � 0.2 ms; and dA off, 2.0 � 0.3 ms).

Interestingly, when the mobility of the mRFP-SC35 pro-tein itself was measured in speckles, the same two mobilityfractions were observed (Figure 3C), although the fastermoving fraction was a somewhat higher percentage of thetotal. A similar distribution of SC35 mobility classes wasobserved when measurements were taken outside a speckle(Figure 3C). Therefore, the behavior of the mRFP-SC35 pro-tein as judged by FCS was similar to the behavior of oli-go(dT), both with respect to the presence of two mobilitycategories, presumably one bound and the other free, and in

Figure 2. In vivo signal distribution and in situ reverse transcrip-tion in the mRFP-SC35 HeLa cell line after uptake of fl-oligo(dT) orfl-oligo(dA). (A and B) HeLa cells stably transfected with SC35-mRFP were allowed to take up fluorescently labeled oligo(dT) andthe distribution of signal was visualized as described in Materialsand Methods. (A) SC35-mRFP. (B) fl-oligo(dT). Bars, 3 �m. (C and D)After uptake of either fl-oligo(dT) or fl-oligo(dA), cells were fixedand subjected to in situ reverse transcription as described in Mate-rials and Methods. Incorporation of biotin-labeled deoxynucleotideswas detected using anti-biotin antibody coupled to horseradishperoxidase as described in text. (C) Bright field image of cellscontaining oligo(dT). (D) Bright field image of cells containing oli-go(dA). Bars, 9.4 �m.

the fact that there was no difference in the mobility of thesefractions on or off speckles.

A low abundance, very slow third component was alsoidentified using FCS in some oligo(dT)-containing cells (Fig-ure 3B, dT white bars). This minor, low-mobility fractionwas not thought to contain hybridized oligo(dT) because itwas also present in some control oligo(dA)-containing cells(Figure 3B, dA white bars). The nature of this slow fractionof oligo(dT) and oligo(dA) is not understood at present, butit should be noted that a slow component with similarcharacteristics is also detected in some cells in which theintranuclear mobility of GFP is measured (Wachsmuth et al.,2000; Baudendistel and Langowski, unpublished observa-tions). Another observation, perhaps related, was that insome FCS measurements an initial bleaching of signal oc-curred. We were not able to characterize this very slowmoving or immobile fraction in detail, but it was detected inboth speckles and in the nucleoplasm at approximatelyequal frequencies.

We also used FCS to measure the mobility of the hybrid-ized oligo(dT) in cells at 22°C compared with 37°C. Ourprevious studies had shown no difference in poly(A) RNAmobility in L6 myoblasts as a function of temperature (Politzet al., 1999) or under conditions that inhibited ATP produc-tion (Politz et al., 1998). However, in these previous studiesit was not possible to distinguish speckles from other areaswithin the nucleoplasm. Using the stable mRFP-SC35 HeLacell line, where oligo(dT) mobility within and outside speck-les could easily be compared at 22 and 37°C, no significantdifference was observed in the shape of the autocorrelationcurves obtained at each temperature (our unpublished data),and the best fit mobility distributions showed no significantdifference in the behavior of the poly(A) RNA at eithertemperature (Figure 3D, red bars). As described above, at37°C it took poly(A) RNA an average of 22 � 5 ms totraverse a confocal volume within a speckle and 20 � 4 msto traverse the same distance in the nucleoplasm proper.When the measurements were made at 22°C instead, it tookpoly(A) RNA an average of 27 � 6 ms to traverse theconfocal volume within a speckle and 28 � 8 ms in thenucleoplasm. Additional details of the FCS measurementsare provided in the Supplemental Material.

Visualization and Tracking of Poly(A) RNA byPhotoactivation in Speckles and NucleoplasmTo visually track the movement of poly(A) RNA in and outof speckles and within the nucleoplasm, we next introducedoligo(dT) labeled with photoactivatable fluorescein (Mitchi-son et al., 1994; Politz, 1999; Politz et al., 1999) to the SC35-expressing HeLa cell line. The coverslips containing thegrowing cells were mounted in a temperature-controlledchamber on an inverted microscope, and the oligo present ina particular speckle was photoactivated using a laser di-rected through a pinhole (Politz et al., 1999, 2004). The un-caging spot within the nucleus was typically 1.0–1.5 �m indiameter (see Figure 6 for image), similar to or smaller thanthe speckles. The movement of the photoactivated signalaway from the speckle was tracked over time by capturingsequential digital images (Figure 4). The signal was ob-served to move out in all directions from the uncaging siteinto the surrounding nucleoplasm but did not enter nucleoli.Signal moved to remote speckles in a manner indistinguish-able from the movement through the nucleoplasm, but withno accumulation of signal in other speckles (see red circledspeckles in Figure 4). There was no evidence of a concentra-tion of signal moving in a directed way toward anotherspeckle or toward the nuclear envelope. Instead, the pattern

Figure 3. Mobility of oligo(dT) and oligo(dA) on and off specklesin mRFP-SC35 HeLa cells measured using FCS. (A) Autocorrelationcurves of cells containing oligo(dT), based on FCS measured eitherwithin a speckle (blue) or in the nucleoplasm (off a speckle, red). (B)Fraction of oligo(dT) and oligo(dA) present in different mobilityclasses measured within speckles (on) and in the nucleoplasm (off).Each kinetic component is indicated by black, red, or open bars inthe histogram. The 10- to 100-ms component (red) was undetectablein oligo(dA) containing-cells. (C) Fraction of mRFP-SC35 proteinpresent in different mobility classes measured within speckles (on)and in the nucleoplasm (off). (D) Average fraction of oligo(dT)present in different mobility classes measured using FCS withinspeckles (on) and in the nucleoplasm (off) at 22 and 37°C.

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of signal dispersion reproducibly approximated a Gaussiandistribution.

The oligo(dT) moved away from the speckle much moreslowly than a control nonhybridizing oligo(dA). Over half ofthe oligo(dA) had left the uncaging site by 2 s, whereas�90% of the oligo(dT) remained and then moved awaymore slowly (Figure 5A). This is the expected result because,as observed in the FCS experiments, the oligo(dT) that ishybridized to poly(A) RNA moves much more slowly thandoes free oligo in the nucleus. The signal intensity along aline drawn through the nucleus and the center of the uncag-ing site generally described a Gaussian distribution (typicalexample in Figure 5B, broken curves), although the curveswere never completely smooth. The small fluctuations insignal distribution along the curve represent regions wherethe signal did not spread in a completely uniform way andthus may reflect the presence of obstacles in the intranuclearlandscape.

When the rate of movement away from the uncaging sitewas plotted as a function of the width (at e2) of the Gauss-ian distribution at increasing times (Cardullo et al., 1991), themean square displacement of the signal was found to belinearly proportional to time (Figure 5C), as expected of adiffusive process. The average estimated diffusion coefficientusing this method was 0.67 � 0.05 �m2/s, which was verysimilar to that estimated earlier in the nucleoplasm of L6cells using the same method (Politz et al., 1999). Because thismethod of estimating a diffusion coefficient is dependent onthe position at which the width of the Gaussian is measured,and also does not account for any potential optical blurring,we also used a more refined algorithm to estimate the dif-fusion coefficient. This algorithm fit the relative shapes of theentire Gaussian distributions over multiple time points witha two-component model (Figure 5B, smooth lines) to moreaccurately estimate a diffusion coefficient (Fogarty et al.,2000; Figure 6). Using this method, an average diffusioncoefficient of 0.32 � 0.04 �m2/s was estimated.

When a site in the nucleoplasm outside the speckle wasuncaged, the signal was observed to behave in a virtuallyidentical manner to signal uncaged in a speckle: it still leftthe site at a similar rate (Figure 5, D and E), and the squareof the displacement again varied linearly with time (Figure5F). The average diffusion coefficient was estimated to be0.39 � 0.04 �m2/s using the global algorithm (Figure 5E,

smooth lines show fit), very similar to the diffusion coeffi-cient estimated within speckles. This result is consistent withthe FCS experiments described above in which no significantdifference was observed in the mobility of poly(A) RNAwithin speckles or in the nucleoplasm.

We next followed the movement of poly(A) RNA afterphotoactivation inside a speckle at 22°C, rather than at 37°C.No change in the characteristics of movement of the taggedpoly(A) RNA was observed (Figure 5, G and H), and theaverage diffusion coefficient of 0.27 � 0.08 �m2/s estimatedfrom the global fit algorithm was not significantly differentfrom that estimated at 37°C (0.32 � 0.04 �m2/s; Figure 5B).No difference was observed in the rate of movement at thetwo temperatures in the nucleoplasm either (our unpub-lished data), as has been reported previously for poly(A)RNA in L6 myoblasts (Politz et al., 1999; Politz and Pederson,2000). Therefore, using either FCS or photoactivation tech-niques, poly(A) RNA showed similar dynamics in the nu-cleoplasm and the speckle, and no evidence was obtainedfor the presence of a distinct slow-moving poly(A) RNApopulation within the speckle.

Although the results presented up to now indicated thatmost poly(A) in speckles was dynamically exchanging withthe nucleoplasm, we wanted to address the degree to whichour methods could have detected a minor population ofimmobile RNA in the speckle because this was the keyhypothesis being tested. Additionally, we wanted to deter-mine whether uncaged signal above and below the plane offocus was affecting our diffusion coefficient estimates. Wetherefore carried out a series of quantitative uncaging sim-ulations. Figure 6 shows simulations (see Materials and Meth-ods) of a population of molecules moving away from a1-�m-diameter uncaging site with a diffusion coefficient of0.3 �m2/s within a nucleus that is 15 �s in diameter underour standard widefield imaging conditions (blurred, Figure6, A and B) versus a simulated ideal case where no opticalblurring of the signal takes place (i.e., contributions fromuncaged molecules above and below the plane of focus wereomitted from the simulation; Figure 6, D and E). When theglobal fit algorithm was used to fit the Gaussian curves forthe blurred simulation at each time point (Figure 6C, smoothlines), the best fit was obtained when a two-componentGaussian spatial profile was assumed. The second compo-nent thus very likely represents the blurred, or out-of-focus

Figure 4. Movement of oligo(dT):poly(A)RNA hybrids away from a speckle after pho-toactivation. mRFP-SC35 HeLa cells were al-lowed to take up caged-fl oligo(dT) and thenthe probe was photoactivated using 360-nmlight directed at a speckle in a live cell nu-cleus as described in Materials and Methods.The uncaging site is marked with a whitecircle in the “caged” panel, and two specklesare circled in red in both the SC35 panel andin the final panel. High-speed time-lapse dig-ital image microscopy was used to capture2D images of the signal as it moved awayfrom the uncaging site. (The brighter dotsvisualized here sometimes occur in cells thathave taken up oligodeoxynucleotides and donot correspond to hybridization sites (Politzand Pederson, unpublished data; also seeLorenz et al., 1998). Bar, 2.6 �m.

light, because it is not present in the fits to the nonblurredimage (Figure 6, E and F). It therefore can be concluded thatthe contribution of the out-of-focus signal above and belowthe plane of focus in our experiments inflates our estimatefor the diffusion coefficient of poly(A) RNA by at most afactor of 2, and we can account for this using the twocomponent global fit algorithm. As mentioned, we used thistwo-component global fit algorithm to calculate the Ds forall the uncaging data described above, so that the contribu-tion of the optical blurring was removed.

We noted that the global fits to the real data Gaussiandistributions shown in Figure 5 do not fit the early timepoints as well as the global fits to the simulations shown inFigure 6 (compare Figure 5C to 6C). We think this is becauseof the presence of free oligo near the uncaging site at theseearly times points. Indeed, the FCS studies described abovepredict that about half of the oligo(dT) in the nucleus inunhybridized, but this fraction cannot be tracked in the timeframe of the uncaging studies because free oligo wouldmove away from the site within the first few time points

Figure 5. Characterization of signal move-ment away from photoactivation site. (A)Probe was uncaged in speckles in cells con-taining either oligo(dT) (red curve) or oli-go(dA) (black curve) and the average signal/pixel remaining at the uncaging site (bleachadjusted) was calculated for each time pointand plotted. The bar on each time point rep-resents the SE of the mean. (B) The Gaussiandistributions of the signal intensity across aline across the nucleus and through the cen-ter of the uncaging site were digitally re-corded at successive times after photoactiva-tion (broken curves, top to bottom, 65, 450,900, 1350, 1800, 3150, and 3500 ms), and aglobal algorithm was used to determine thebest fit diffusion coefficient for the curves andtime simultaneously (see Figure 6; see Mate-rials and Methods). A representative distribu-tion and the simulated fit (smooth lines) foran uncaging on a speckle is shown here; thediffusion coefficient estimated from this un-caging was 0.346 �m2/s. (C) The meansquare displacement (at e2) versus timeplotted for the same uncaging site as shownin B. The line through the points is based ona linear least squares regression analysis(R2 � 0.92) and predicts a diffusion coefficientof 0.7 �m2/s. (D) Average signal remainingat uncaging sites over time after uncaging on(red curve) or off (blue curve) a speckle incells containing oligo(dT). Error bars for eachpoint have been omitted so that the twocurves can be seen clearly. For each point theSE of the mean was less than or equal to�3%. (E) Same as in B except the uncagingsite was nucleoplasmic (off a speckle). Thediffusion coefficient calculated from thisglobal fit is 0.285 �m2/s. (F) The mean squaredisplacement (at e2) versus time plotted forthe same uncaging site as shown in E. (D �0.6 �m2/s; R2 � 0.98). (G) Average fraction ofsignal remaining at uncaging site after pho-toactivation of caged-fl oligo(dT) in specklesat 37°C (pink) and 22°C (blue). The bar oneach point is the SE of the mean. (H) Same asB except at 22°C. The diffusion coefficientestimated from this global fit is 0.351 �m2/s.

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(seconds). This interpretation is supported by the fact that ifthe global fits are done only to the first two time points afteruncaging, a faster diffusion coefficient is obtained.

Finally, we built differing levels of a putative immobileRNA population into the simulation as fixed parameters todetermine what fraction of immobile molecules would bedetectable in the photoactivation-tracking experiments.These simulations revealed that an immobile RNA popula-tion comprising as little as 5% of the total signal within theuncaging site would have been easily visualized in boththe tracking images (compare Figure 6G with H) and in theGaussian plots of the intensity distribution used to estimatethe diffusion coefficient (compare Figure 6I with B).

DISCUSSION

In this work, we have found that the mobility of poly(A)RNA in HeLa cells is essentially the same both in specklesand in the nucleoplasm. Therefore, although this RNA pop-ulation includes not only processed mRNA destined forcytoplasmic export but also a substantial fraction of nucleus-retained poly(A) RNA molecules (Perry et al., 1974, Hermanet al., 1976; Huang et al., 1994; Morey and Avner, 2004) thatmight have been thought to be involved in maintainingnuclear substructure in some way, we did not find anypoly(A) RNA to be specifically tethered within speckles. Ourresults do not rule out the presence of a very small slow-moving or immobile class of poly(A) RNA in the nucleus,

but if it exists, our results indicate that it is �5% of the totalpoly(A) RNA and is present in similar amounts in bothspeckles and the nucleoplasm. Therefore, it is unlikely thatpoly(A) RNA serves as an immobile scaffolding to form thespeckle; rather, it can move freely in and out of speckles inall directions and visit other speckles.

These findings are consistent with numerous studies onthe mobilities of nuclear proteins that have demonstratedrapid exchange between the nucleoplasm and speckles(Kruhlak et al., 2000; Phair and Misteli, 2000; Molenaar etal., 2004), the nucleolus (Phair and Misteli, 2000; Snaar et al.,2000; Chen and Huang, 2001), and Cajal bodies (Snaar et al.,2000; Handwerger et al., 2003; Dundr et al., 2004). We alsoshow here that the SC35 protein is extremely dynamic inboth within and outside speckles. It is therefore becomingincreasingly clear that nuclear proteins that were oncethought to perhaps serve static structural roles because theyseemed concentrated in a certain structure, actually move inand out of these structures very rapidly (Misteli, 2001; Bubu-lya and Spector, 2004). Our results demonstrate that poly(A)RNA behaves in a similar manner and exchanges rapidlybetween speckles and the nucleoplasm.

We looked for temperature-dependent behavior of thepoly(A) RNA movement in speckles, but we observed nodifference in the rate of movement of poly(A) RNA at 22versus 37°C within speckles or in the nucleoplasm. If move-ment into or out of speckles were dependent on enzymaticactivity (perhaps requiring a motor protein driven by ATP

Figure 6. Simulations of a population ofmolecules moving away from a one microndiameter uncaging site with a diffusion coef-ficient of 0.3 �m2/s. (A) Simulated (see Mate-rials and Methods) wide-field microscope im-age of the uncaged spot before any diffusionhas occurred. The uncaged profile is reim-aged with both in-focus (the aperture) andout-of-focus components. (B) Plot of intensi-ties along a line through the center of the spotin A (circles), are well fit by a two componentGaussian model (black line) compared with asingle Gaussian model (red line). The twoGaussian components capture the in-focus(narrower) and out-of-focus (broader) contri-butions (see Materials and Methods). (C) Plot ofintensities along the same line over time, afterallowing the initial uncaged distributionshown in A and B to diffuse in 3-D with D �0.3 �m2/s. Colors (black, red, green, cyan,blue, magenta, violet, and orange) correspondto data from images at times 0.15, 0.6, 1.05,1.5, 1.95 (2.4 and 2.85 not shown), 3.3, 3.75,and 8.25 s, respectively. The intensity linedata from the images up to 3.75 s were jointlyfit with the equation for a single (Gaussian)diffusion component convolved with a twoGaussian component initial uncaged distribu-tion (solid lines). The diffusion coefficientfrom the fit was D � 0.38 �m2/s. (D) Same asin A except before being reimaged (blurred)by the PSF of the microscope. This yields onlythe infocus portion of the 3-D uncaged spot.(E) Plot of intensities along a line through the

center of D (circles) are well fit by a one-component gaussian model (black line). (F) Same as C except the lines were through D, and theequation fit to the data is a single diffusion component convolved with a single Gaussian component for the initial distribution. From the fit,D � 0.34 �m2/s. (G) From the same simulation as in A, after diffusing for 8.25 s (orange circles in C). (H) Same as in G except for thissimulation, 5% of the uncaged molecules at the uncaging spot were assumed to be immobile. (I) Same as C, but the line was drawn throughthe center of the uncaging site on H. The presence of the 5% fixed molecules within a 2 �m speckle is quite evident by 8 s (orange circles,compare with C). Bars (A, C, F and G), 4 �m.

hydrolysis, for example), one would expect to see a three- tofivefold slowing of the rate at the lower temperature becauseenzymatic reactions have Q10s (change in reaction rate withevery 10 K change in temperature) between 2 and 3. Fur-thermore, if poly(A) were being directed specifically to thenuclear pores by a metabolic energy-requiring process, onewould expect to see a change in the distribution of thepoly(A) RNA as it moved out into the nucleoplasm from thespeckle at the different temperatures. However, we did notsee this. Thus, the simplest interpretation is that poly(A)RNA can move freely between speckles and the nucleo-plasm of HeLa cells without a direct input of metabolicenergy.

Calapez et al. (2002) reported an �1.5- to 2-fold (estimatedfrom Figure 8 in Calapez et al., 2002) reduction in the rate ofmovement (which gives a threefold reduction in diffusioncoefficient) of two different GFP-labeled proteins bound topoly(A) RNA at 37 versus 22°C and suggested that thisimplies metabolic energy-dependent mobility. Similarly,Molenaar et al. (2004) have reported an �1.5-fold (estimatedfrom their Figure 7B) decrease in the rate of recovery afterphotobleaching of a tagged poly(A) RNA at 37 versus 22°Cand suggested on this basis that metabolic energy is neces-sary for poly(A) RNA mobility. However, reductions of thismagnitude are not easily explained by rate decreases inenzymatic reactions because, as explained above, one wouldexpect to see a much larger effect upon a 15°C temperatureshift (see also Shav-Tal et al., 2004 for a discussion of thispoint). In addition, a recent study of mRNA movement inthe nucleus of mammalian cells has somewhat clarified thesituation by revealing that ATP is required for the resump-tion of movement when RNA becomes corralled within tightconfinements, rather than the nucleotide being involved inthe movement itself (Vargas et al., 2005).

Molenaar et al. (2004) also reported that several poly(A)binding proteins moved in and out of speckles at ratessimilar to those reported here for both bound SC35 andpoly(A) RNA, and by others for RNA-binding proteins(Phair and Misteli, 2000; Misteli, 2001; Calapez et al., 2002),but they reported a 5- to 10-fold slower diffusion coefficientfor poly(A) RNA itself. It is unclear, however, whether the2�-O-methyl modified oligo(U) probe used by Molenaar et al.(2004) was actually hybridized to poly(A). Hybridizationwas tested in only one way: U2OS cells were treated with thecytotoxic drug cordycepin for 16 h, and after this treatmenttime, it was observed that newly injected 2�-O-methyl oli-go(U) was more mobile within the nucleus and no longeraccumulated in speckles. Because cordycepin interferes withpolyadenylation, it was concluded that the probe must havebeen hybridized in the untreated cells. However, cordycepinhas not been used in previous studies on mammalian cellnuclear RNA metabolism for more than 1–3 h (Siev et al.,1969; Penman et al., 1970; Darnell et al., 1971; Mendecki et al.,1972) because of severe toxicity effects. Moreover, it has beenclearly established that cordycepin inhibits the productionof cytoplasmic mRNA in HeLa cells within 25–40 min (Pen-man et al., 1970). The mRNA population of HeLa cells con-sists of at least two kinetic components; 30–40% has anaverage half-life of 2–7 h, and the rest has an average half-life of 24 h (Singer and Penman, 1973; Puckett and Darnell,1977). A 16-h treatment with cordycepin would thereforereduce the shorter lived mRNA population to 6–25% of itsoriginal concentration; cells treated in this way may notreflect the behavior of normal cells.

We observed one more interesting property of poly(A)RNA in the experiments described here: poly(A) RNA canvisit more than one speckle as it travels throughout the

nucleoplasm. This implies that a visit to a speckle does notchange the characteristics of the poly(A) RNA in such a waythat it is unable to pass through other speckles. A similarphenomenon was observed in live cell tracking of 28S rRNA,which was observed to move between nucleoli (Politz et al.,2003). Certain nuclear proteins have also been observed tomove between nuclear compartments, such as fibrillarinbetween nucleoli and SF2/ASF between speckles (Kruhlak etal., 2000; Phair and Misteli, 2000), but these proteins are notdestined for transport to the cytoplasm, as is the aforemen-tioned 28S rRNA and polyadenylated mRNA. It thereforeseems that even macromolecules that must be transported tothe cytoplasm can roam freely not only throughout thenucleoplasm but also enter and exit similar nuclear compart-ments with no direct requirement of metabolic energy.

ACKNOWLEDGMENTS

We thank Supriya Prasanth for help in Western blot analysis. This work wassupported by National Institutes of Health Grants GM-60551 to J. P., R. T. andT. P.; GM-42694 to D. S.; and DK-32520 to K. F.; National Science FoundationGrants DB19200027 and DB19724611 to the University of Massachusetts Bio-medical Imaging Facility; and a grant to J. L. from the Volkswagen Founda-tion as part of the program “Physics, Chemistry, and Biology with SingleMolecules.”

REFERENCES

Bubulya, P. A., and Spector, D. L. (2004). “On the move”ments of nuclearcomponents in living cells. Exp. Cell Res. 296, 4–11.

Calapez, A., Pereira, H. M., Calado, A., Braga, J., Rino, J., Carvalho, C.,Tavanez, J. P., Wahle, E., Rosa, A. C., and Carmo-Fonseca, M. (2002). Theintranuclear mobility of messenger RNA binding proteins is ATP dependentand temperature sensitive. J. Cell Biol. 159, 795–805.

Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S.,Zacharias, D. A., and Tsien, R. Y. (2002). A monomeric red fluorescent protein.Proc. Natl. Acad. Sci. USA 11, 7877–7882.

Cardullo, R. A., Mungovan, R. M., and Wolf, D. E. (1991). Imaging membraneorganization and dynamics. In: Biophysical and Biochemical Aspects of Flu-orescence Spectroscopy, ed. T. D. Dewey, New York: Plenum Publishing,231–260.

Carrington, W. A., Lynch, R. M., Moore, E. D., Isenberg, G., Fogarty, K. E., andFay, F. S. (1995). Superresolution three-dimensional images of fluorescence incells with minimal light exposure. Science 268, 1483–1487.

Carter, K. C., Bowman, D., Carrington, W., Fogarty, K., McNeil, J. A., Fay,F. S., and Lawrence, J. B. (1993). A three-dimensional view of precursormessenger RNA metabolism within the mammalian nucleus. Science 259,1330–1335.

Chen, D., and Huang, S. (2001). Nucleolar components involved in ribosomebiogenesis cycle between the nucleolus and nucleoplasm in interphase cells.J. Cell Biol. 153, 169–176.

Cmarko, D., Verschure, P. J., Martin, T. E., Dahmus, M. E., Krause, S., Fu,X. D., van Driel, R., and Fakan, S. (1999). Ultrastructural analysis of transcrip-tion and splicing in the cell nucleus after bromo-UTP microinjection. Mol.Biol. Cell 10, 211–223.

Darnell, J. E., Philipson, L., Wall, R., and Adesnik, M. (1971). Polyadenylicacid sequences: role in conversion of nuclear RNA into messenger RNA.Science 174, 507–510.

Deerinck, T. J., Martone, M. E., Lev-Ram, V., Green, D. P., Tsien, R. Y., Spector,D. L., Huang, S., and Ellisman, M. H. (1994). Fluorescence photooxidationwith eosin: a method for high resolution immunolocalization and in situhybridization detection for light and electron microscopy. J. Cell Biol. 126,901–910.

Dundr, M., Hebert, M. D., Karpova, T. S., Stanek, D., Xu, H., Shpargel, K. B.,Meier, U. T., Neugebauer, K. M., Matera, A. G., and Misteli, T. (2004). In vivokinetics of Cajal body components. J. Cell Biol. 164, 831–842.

Eberwine, J., Spencer, C., Miyashiro, K., Mackler, S., and Finnell, R. (1992).Complementary DNA synthesis in situ: methods and applications. MethodsEnzymol. 216, 80–100.

Eils, R., Gerlich, D., Tvarusko, W., Spector, D. L., and Misteli, T. (2000).Quantitative imaging of pre-mRNA splicing factors in living cells. Mol. Biol.Cell 11, 413–418.

Vol. 17, March 2006 1247

Fakan, S., and Nobis, P. (1978). Ultrastructural localization of transcriptionsites and of RNA distribution during the cell cycle of synchronized CHO cells.Exp. Cell Res. 113, 327–337.

Fakan, S., Leser, G., and Martin, T. E. (1984). Ultrastructural distribution ofnuclear ribonucleoproteins as visualized by immunocytochemistry on thinsections. J. Cell Biol. 98, 358–363.

Fogarty, K. E., Kidd, J. F., Tuft, R. A., and Thorn, P. (2000). Mechanismsunderlying InsP3-evoked global Ca2� signals in mouse pancreatic acinar cells.J. Physiol. 526, 515–526.

Guillot, P. V., Xie, S. Q., Hollinshead, M., and Pombo, A. (2004). Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in thenucleus of human cells. Exp. Cell Res. 295, 460–468.

Handwerger, K. E., Murphy, C., and Gall, J. G. (2003). Steady-state dynamicsof Cajal body components in the Xenopus germinal vesicle. J. Cell Biol. 160,495–504.

Helfman, D. M., Ricci, W. M., and Finn, L. A. (1988). Alternative splicing oftropomyosin pre-mRNAs in vitro and in vivo. Genes Dev. 2, 1627–1638.

Herman, R. C., Williams, J. G., and Penman, S. (1976). Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclearRNA of HeLa cells. Cell 7, 429–437.

Huang, S., and Spector, D. L. (1991). Nascent pre-mRNA transcripts areassociated with nuclear regions enriched in splicing factors. Genes Dev. 5,2288–2302.

Huang, S., Deerinck, T. J., Ellisman, M. H., and Spector, D. L. (1994). In vivoanalysis of the stability and transport of nuclear poly(A)� RNA. J. Cell Biol.126, 877–899.

Huang, S., and Spector, D. L. (1996a). Dynamic organization of pre-mRNAsplicing factors. J. Cell Biochem. 62, 191–197.

Huang, S., and Spector, D. L. (1996b). Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription. J. Cell Biol. 133, 719–732.

Jimenez-Garcia, L. F., and Spector, D. L. (1993). In vivo evidence that tran-scription and splicing are coordinated by a recruiting mechanism. Cell 73,47–59.

Johnson, C., Primorac, D., McKinstry, M., McNeil, J., Rowe, D., and Lawrence,J. B. (2000). Tracking COL1A1 RNA in osteogenesis imperfecta: splice-defec-tive transcripts initiate transport from the gene but are retained within theSC35 domain. J. Cell Biol. 150, 417–432.

Kim, S. A., and Schwille, P. (2003). Intracellular applications of fluorescencecorrelation spectroscopy: prospects for neuroscience. Curr. Opin. Neurobiol.13, 583–590.

Kish, V. M., and Pederson, T. (1977). Heterogeneous nuclear RNA secondarystructure: oligo(U) sequences base-paired with poly(A) and their possible roleas binding sites for heterogeneous nuclear RNA-specific proteins. Proc. Natl.Acad. Sci. USA 74, 1426–1430.

Krichevsky, O., and Bonnet, G. (2002). Fluorescence-correlation spectroscopy:the technique and its applications. Rep. Prog. Phys. 65, 251–297.

Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E., Bazett-Jones, D. P., andHendzel, M. J. (2000). Reduced mobility of the alternate splicing factor (ASF)through the nucleoplasm and steady state speckle compartments. J. Cell Biol.150, 41–51.

Lamond, A. I., and Spector, D. L. (2003). Nuclear speckles: a model for nuclearorganelles. Nat. Rev. 4, 605–612.

Lorenz, P., Baker, B. F., Bennett, C. F., and Spector, D. L. (1998). Phosphoro-thioate antisense oligonucleotides induce the formation of nuclear bodies.Mol. Biol. Cell 9, 1007–1023.

Magde, D., Elson, E. L., and Webb, W. W. (1972). Thermodynamic fluctuationsin a reacting system: measurement by fluorescence correlation spectroscopy.Phys. Rev. Lett. 29, 705–708.

Mattaj, I. (1994). Splicing in space. Nature 372, 727–728.

Mendecki, J., Lee, S. Y., and Brawerman, G. (1972). Characteristics of thepolyadenylic acid segment associated with messenger ribonucleic acid inmouse sarcoma 180 ascites cells. Biochemistry 11, 792–798.

Misteli, T. (2001). Protein dynamics: implications for nuclear architecture andgene expression. Science 291, 843–847.

Misteli, T., Caceres, J. F., Clement, J. Q., Krainer, A. R., Wilkinson, M. F., andSpector, D. L. (1998). Serine phosphorylation of SR proteins is required fortheir recruitment to sites of transcription in vivo. J. Cell Biol. 143, 297–307.

Misteli, T., Caceres, J. F., and Spector, D. L. (1997). The dynamics of apre-mRNA splicing factor in living cells. Nature 387, 523–527.

Misteli, T., and Spector, D. L. (1996). Serine/threonine phosphatase 1 modu-lates the subnuclear distribution of pre-mRNA splicing factors. Mol. Biol. Cell7, 1559–1572.

Misteli, T., and Spector, D. L. (1999). RNA Polymerase II targets pre-mRNAsplicing factors to transcription sites in vivo. Mol. Cell 3, 697–705.

Mitchison, T. J., Sawin, K. E., and Theriot, J. A. (1994). Caged fluorescentprobes for monitoring cytoskeleton dynamics. In: Cell Biology: A LaboratoryHandbook, Vol. 3, ed. J. E. Celis, New York: Academic Press, 65–74.

Modos, K., Galantai, R., Bardos-Nagy, I., Wachsmuth, M., Toth, K., Fidy, J.,and Langowski, J. (2004). Maximum-entropy decomposition of fluorescencecorrelation spectroscopy data: application to liposome-human serum albuminassociation. Eur. Biophys. J. 33, 59–67.

Molenaar, C., Abdulle, A., Gena, A., Tanke, H. J., and Dirks, R. W. (2004).Poly(A) RNAs roam the cell nucleus and pass through speckle domains intranscriptionally active and inactive cells. J. Cell Biol. 165, 191–202.

Morey, C., and Avner, P. (2004). Employment opportunities for non-codingRNAs. FEBS Lett. 567, 27–34.

Neugebauer, K. M., and Roth, M. B. (1997). Distribution of pre-mRNA splic-ing factors at sites of RNA polymerase II transcription. Genes Dev. 11,3279–3285.

Pederson, T. (1999). Movement and localization of RNA in the cell nucleus.FASEB J. 2, S238–242.

Penman, M., Rosbash, M., and Penman, S. (1970). Messenger and heteroge-neous nuclear RNA in HeLa cells: differential inhibition by cordycepin. Proc.Natl. Acad. Sci. USA 67, 1878–1885.

Perry, R. P., Kelley, D. E., and LaTorre, J. (1974). Synthesis and turnover ofnuclear and cytoplasmic polyadenylic acid in mouse L cells. J. Mol. Biol. 82,315–331.

Phair, R. D., and Misteli, T. (2000). High mobility proteins in the mammaliancell nucleus. Nature 404, 604–609.

Politz, J. C. (1999). Use of caged fluorochromes to track macromolecularmovement in living cells. Trends Cell Biol. 9, 284–287.

Politz, J. C., Browne, E. S., Wolf, D. E., and Pederson, T. (1998). Intranucleardiffusion and hybridization state of oligonucleotides measured by fluores-cence correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95, 6043–6048.

Politz, J. C., and Pederson, T. (2000). Movement of mRNA from transcriptionsite to nuclear pores. J. Struct. Biol. 129, 252–257.

Politz, J. C., and Singer, R. H. (1999). In situ reverse transcription for thedetection of hybridization between oligonucleotides and their intracellulartargets. Methods 18, 281–285.

Politz, J. C., Taneja, K. L., and Singer, R. H. (1995). Characterization ofhybridization between synthetic oligodeoxynucleotides and RNA in livingcells. Nucleic Acids Res. 23, 4946–4953.

Politz, J.C.R., Tuft, R. A., and Pederson, T. (2003). Diffusion-based transport ofnascent ribosomes in the nucleus. Mol. Biol. Cell 14, 4805–4812.

Politz, J.C.R., Tuft, R. A., and Pederson, T. (2004). Photoactivation-basedlabeling and in vivo tracking of RNA molecules in the nucleus. In: Live CellImaging: A Laboratory Manual, ed. D. L. Spector and R. D. Goldman, ColdSpring Harbor, NY: Cold Spring Harbor Laboratory Press, 177–185.

Politz, J. C., Tuft, R. A., Pederson, T., and Singer, R. H. (1999). Movement ofnuclear poly(A) RNA throughout the interchromatin space in living cells.Curr. Biol. 9, 285–291.

Politz, J. C., Yarovoi, S., Kilroy, S. M., Gowda, K., Zwieb, C., and Pederson, T.(2000). Signal recognition particle components in the nucleolus. Proc. Natl.Acad. Sci. USA 97, 55–60.

Prasanth, K. V., Sacco-Bubulya, P. A., Prasanth, S. G., and Spector, D. L.(2003). Sequential entry of components of the gene expression machinery intodaughter nuclei. [erratum appears in Mol. Biol. Cell (2003) 14 following 1743].Mol. Biol. Cell 14, 1043–1057.

Press, W. H., Teukolsky, S. A., Vetterling, W. T., and Flannery, B. P. (1992).Numerical Recipes in C: The Art of Scientific Computing, Cambridge: Cam-bridge University Press.

Puckett, L., and Darnell, J. E. (1977). Essential factors in the kinetic analysis ofRNA synthesis in HeLa cells. J. Cell Physiol. 90, 521–534.

Shav-Tal, Y., Darzacq, X., Shenoy, S. M., Fusco, D., Janicki, S. M., Spector,D. L., and Singer, R. H. (2004). Dynamics of single mRNPs in nuclei of livingcells. Science 304, 1797–1800.

Shopland, L. S., Johnson, C. V., Byron, M., McNeil, J., and Lawrence, J. B.(2003). Clustering of multiple specific genes and gene-rich R-bands aroundSC-35 domains: evidence for local euchromatic neighborhoods. J. Cell Biol.162, 981–990.

Siev, M., Weinberg, R., and Penman, S. (1969). The selective interruption ofnucleolar RNA synthesis in HeLa cells by cordycepin. J. Cell Biol. 41, 510–520.

Singer, R. H., and Penman, S. (1973). Messenger RNA in HeLa cells: kineticsof formation and decay. J. Mol. Biol. 78, 321–334.

Singh, O. P., Bjorkroth, B., Masich, S., Wieslander, L., and Daneholt, B. (1999).The intranuclear movement of Balbiani ring premessenger ribonucleoproteinparticles. Exp. Cell Res. 251, 135–146.

Snaar, S., Wiesmeijer, K., Jochemsen, A. G., Tanke, H. J., and Dirks, R. W.(2000). Mutational analysis of fibrillarin and its mobility in living human cells.J. Cell Biol. 151, 653–662.

Ueno, Y., Kumagai, I., Haginoya, N., and Matsuda, A. (1997). Effects of5-(N-aminohexyl)carbamoyl-2�deoxyuridine on endonuclease stability andthe ability of oligodeoxynucleotide to activate RNase H. Nucleic Acids Res.25, 3777–3782.

Vargas, D. Y., Raj, A., Marras, S.A.E., Kramer, F. R., and Tyagi, S. (2005).Mechanism of mRNA transport in the nucleus. Proc. Natl. Acad. Sci. USA 102,17008–17013.

Wachsmuth, M., Waldeck, W., and Langowski, J. (2000). Anomalous diffusionof fluorescent probes inside living cell nuclei investigated by spatially re-solved fluorescence correlation spectroscopy. J. Mol. Biol. 298, 677–689.

Wachsmuth, M., Weidemann, T., Muller, G., Hoffmann-Rohrer, U. W., Knoch,T. A., Waldeck, W., and Langowski, J. (2003). Analyzing intracellular bindingand diffusion with continuous fluorescence photobleaching. Biophys. J. 84,3353–3363.

Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, R.,and de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals transcrip-tion by RNA polymerase II in domains scattered throughout the nucleus.J. Cell Biol. 122, 283–293.

Zeng, C., Kim, E., Warren, S. L., and Berget, S. M. (1997). Dynamic relocationof transcription and splicing factors dependent upon transcriptional activity.EMBO J. 16, 1401–1412.

Zhang, G., Taneja, K. L., Singer, R. H., and Green, M. R. (1994). Localizationof pre-mRNA splicing in mammalian nuclei. Nature 372, 809–812.

ZhuGe, R., Fogarty, K. E., Tuft, R. A., Lifshitz, L. M., Sayer, K., and Walsh, J. V.(2000). A novel method for direct measurement of Ca2� spark signal massreveals dynamics of signaling between ryanodine receptors and Ca2�-acti-vated K� channels in smooth muscle. J. Gen. Physiol. 116, 845–864.

Zou, H., Lifshitz, L. M., Tuft, R. A., Fogarty, K. E., and Singer, J. J. (1999).Imaging Ca2� entering the cytoplasm through a single opening of a plasmamembrane cation channel. J. Gen. Physiol. 114, 575–588.

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Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

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